EA033572B1 - Benzimidazole compounds as jak1 inhibitors - Google Patents

Abstract

The present invention relates to certain benzimidazole compounds or pharmaceutically acceptable salts thereof, that inhibit Janus kinase 1 (JAK1), pharmaceutical compositions comprising the compounds, and methods of using the compounds to treat certain types of cancer.

Description

The present invention relates to certain benzimidazole compounds or their pharmaceutically acceptable salts that inhibit Janus kinase 1 (JAK1), to pharmaceutical compositions containing said compounds, and to methods of using said compounds to treat certain types of cancer.

JAK kinases are a family of tyrosine kinases that regulate the tyrosine phosphorylation of various effectors and initiate the activation of downstream signaling pathways. JAK1 is a member of this family that mediates the activation of the signal transducer and transcription activator 3, STAT3. Continuous activation of STAT3 causes the formation of tumors and promotes the survival and proliferation of cancer cells. Aberrations in STAT3 activation have been shown to destroy the tumor's immune surveillance in the tumor microenvironment. Therefore, inhibition of JAK1 can block the activation of STAT3, which leads to suppression of tumor growth and immune surveillance of the tumor. In addition, the activation of JAK1 mutations was identified in acute lymphoblastic leukemia of T-line cells and in hepatocellular carcinoma of Asian residents, and its oncogenicity was shown. The results suggest that JAK1 is a possible cancer target.

JAK2 is known to form homodimers that mediate signaling along the path from EPO and TPO receptors to STAT5, which regulates the production of red blood cells and platelets. Inhibition of JAK2 can lead to anemia and thrombocytopenia. However, JAK1 does not exhibit such activity and, therefore, it can be assumed that compounds that selectively inhibit JAK1 may have an improved hematotoxicity and / or immunogenicity profile than compounds that selectively inhibit JAK2 or double JAK1 / 2 inhibitors.

JAK kinase inhibitor compounds are described in the literature. For example, in US 2015/0203455, certain benzimidazole compounds that are JAK inhibitors are described.

There remains a need for alternative JAK1 inhibitors for the treatment of cancer. In addition, there remains a need to provide selective JAK1 inhibitors that reduce or prevent JAK2 inhibition. Accordingly, the present invention provides some JAK1 inhibitors that may be useful in treating cancer. In addition, the present invention provides some selective JAK1 inhibitors that can reduce JAK2 inhibition.

The present invention provides a compound of formula I

F ₃ C he ^ NH

CH3 or a pharmaceutically acceptable salt thereof.

Preferably, the present invention provides a compound selected from the group consisting of (2S) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1- methyl-1H-benzimidazole-6yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

CH ₃ or a pharmaceutically acceptable salt thereof and (2I) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole -6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

- 1,033572

or a pharmaceutically acceptable salt thereof.

Preferably, the present invention provides a compound of (2S) -3 {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole -6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

CH ₃ or a pharmaceutically acceptable salt thereof.

More preferably, the present invention provides a compound representing (2R) -3- {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H -benzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

or a pharmaceutically acceptable salt thereof.

As a specific embodiment, the present invention provides a compound representing (2R) -3- {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1- methyl-1H-benzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol.

As a specific embodiment, the present invention also provides a compound representing (2S) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1 -methyl-1Nbenzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol.

The present invention provides a pharmaceutical composition comprising (2K) -3- {| cis-4 ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole- 6-yl} oxy) cyclohexyl] amino} 1,1,1-trifluoropropan-2-ol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient. The present invention provides a pharmaceutical composition comprising (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl

- 2 033572

1H-benzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention provides a method of treating cancer, comprising administering to a patient in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. The present invention provides a method of treating cancer, comprising administering to a patient in need thereof an effective amount of (2I) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3yl) amino] -1-methyl-1H-benzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol or a pharmaceutically acceptable salt thereof. The present invention provides a method of treating cancer, comprising administering to a patient in need thereof an effective amount of (2I) -3 - {[cis-4 - ({4 - [(1,5dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazol-6-yl} oxy) cyclohexyl] amino} -1,1,1 trifluoropropan-2-ol.

The present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in therapy. The present invention provides (2I) -3 - {[cis-4 - ({4 - [(1,5dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazol-6-yl} oxy ) cyclohexyl] amino} -1,1,1 trifluoropropan-2-ol or a pharmaceutically acceptable salt thereof for use in therapy. The present invention provides (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazol-6-yl } hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.

The present invention provides a compound of formula I for use in therapy. The present invention also provides (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1methyl-1H-benzimidazol-6-yl} hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol for use in therapy. The present invention provides (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3yl) amino] -1-methyl-1H-benzimidazol-6-yl} oxy ) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol for use in the treatment of cancer.

The present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer. The present invention provides the use of (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1Hbenzimidazol-6-yl} hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol or its pharmaceutically acceptable salt in the manufacture of a medicament for the treatment of cancer. The present invention also provides the use of (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1Hbenzimidazol-6-yl } hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol in the manufacture of a medicament for the treatment of cancer.

The present invention provides the free base of (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1Npyrazol-3-yl) amino] -1-methyl-1H-benzimidazol-6-yl } hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropane-2ol in crystalline form. The present invention also provides the free base (2R) -3 {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6 -yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol in crystalline form, characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ ± 0.2 at 19.5 ° C combinations with one or more peaks selected from the group consisting of 11.9 °, 15.4 °, and 17.6 °.

The present invention provides a salt of (2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3yl) amino] -1-methyl-1H-benzimidazol-6-yl} hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol with dimethanesulfonic acid in crystalline form. The present invention also provides a salt of (2R) -3- {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6 -yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol with dimethanesulfonic acid in crystalline form, characterized by an X-ray powder diffraction diagram having characteristic peaks expressed in degrees 2θ ± 0.2 ° at 21 , 7 ° in combination with one or more peaks selected from the group consisting of 21.2 °, 18.0 ° and 15.7 °.

In addition, the present invention provides preferred embodiments of the methods and applications described herein in which the cancer is selected from the group consisting of lung cancer, including non-small cell lung cancer, small cell lung cancer and lung adenocarcinoma, adenocarcinoma, hepatocellular carcinoma, including hepatocellular carcinoma carcinoma of Asian residents, colorectal cancer, breast cancer, lymphoma and leukemia, including acute lymphocytic leukemia and acute lymphoblastic leukemia of T-cell cells. Preferred cancers are lung cancer, including non-small cell lung cancer and adenocarcinoma of the lung, adenocarcinoma, hepatocellular carcinoma, including hepatocellular carcinoma of Asian residents, colorectal cancer, breast cancer and leukemia, including acute lymphocytic leukemia. More preferred cancers are non-small cell lung cancer, adenocarcinoma of the lung, breast cancer, and hepatocellular carcinoma in Asia.

It should be understood that the following terms mentioned above and throughout the text of the present invention, unless otherwise indicated, have the following meanings.

- 3 033572

A pharmaceutically acceptable carrier, diluent or excipient is a medium conventional in the art for delivering biologically active agents to mammals, for example humans.

Pharmaceutically acceptable salts or pharmaceutically acceptable salt refers to the relatively non-toxic inorganic and organic salts or salts of the compounds of the present invention.

An effective amount means an amount of a compound or a pharmaceutically acceptable salt thereof according to the present invention or a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to the present invention, which elicits a biological or medical response or a desired therapeutic effect in a tissue, system, animal, mammal or human body, observed by a researcher, veterinarian, doctor or other clinician.

The terms treatment, treat, treating, etc. include slowing or reversing the development of the disorder. These terms also include alleviating, improving, reducing, eliminating, or alleviating one or more symptoms of the disorder or pathological condition, even if the specified disorder or pathological condition is not actually resolved and even if the progression of the disorder or pathological condition is not in itself slowed or reversed.

The compound of the present invention may react, for example, with numerous inorganic and organic acids to form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts and general procedures for their preparation are known in the art. See, for example, P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE (VCHA / Wiley-VCH, 2002); S.M. Berge, et al., Pharmaceutical Salts, Journal of Pharmaceutical Sciences, T. 66, No. 1, January 1977.

The compound of the present invention is preferably formulated as a pharmaceutical composition using a pharmaceutically acceptable carrier, diluent or excipient, and administered in various ways. Preferably, said compositions are for oral administration. Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (A. Gennaro, et al., Ed., 21st ed., Mack Publishing Co., 2005).

The actual amount administered of the compound of the present invention is determined by the physician, taking into account relevant circumstances, including the pathological condition to be treated, the chosen route of administration, the actual compound of the present invention, age, body weight and response of the individual patient, as well as the severity of the patient's symptoms. Daily doses typically range from about 50 to 1000 mg per day, preferably from 80 to 600 mg per day, most preferably 300 mg per day. In some cases, dose levels that are less than the lower limit of the above range may be more appropriate, and in other cases, higher doses may be used. Dose levels can be determined by a person skilled in the art.

The compound of the present invention or a pharmaceutically acceptable salt thereof can be prepared by numerous methods known in the art, as well as the methods described below in the Production Methods and Examples section. The specific synthesis steps of each of the described methods can be combined in various ways to produce a compound of the present invention or a pharmaceutically acceptable salt thereof.

Reagents and starting materials are usually readily available to specialists in this field of technology. Other reagents can be obtained by standard methods of organic and heterocyclic chemistry, methods known to specialists in this field of technology, and the methods described below in the Examples section, including all new methods. The present invention is further illustrated below in the Production Methods and Examples section. Unless otherwise indicated, the names and numbers of the compounds provided herein were obtained using IUPACNAME ACDLABS.

Individual isomers, enantiomers or diastereomers can be isolated or separated by those skilled in the art at any convenient point in the synthesis of compounds using methods such as selective crystallization technology or chiral chromatography (see, for example, Enantiomers, Racemates, and Resolutions (J. Jacques , et al., John Wiley and Sons, Inc., 1981)).

Those skilled in the art will recognize that the compound of the present invention contains at least one chiral center. The present invention includes all individual enantiomers or diastereomers, as well as mixtures of enantiomers and diastereomers of these compounds, including racemates. Preferably, the compound of the present invention exists as a single enantiomer or diastereomer. A single enantiomer or diastereomer can be prepared using chiral reagents or by stereoselective or stereospecific synthesis techniques. Alternatively, a single enantiomer or diastereomer can be isolated from mixtures by standard chiral chromatography or crystallization techniques.

The compound according to the present invention can be obtained in accordance with methods

- 4,033,572 synthesis, well known and accepted in the art. Suitable reaction conditions for the steps of these reactions are well known in the art, and suitable substitutions for solvents and co-reactants are known to those skilled in the art. In the same way, it will be understood by those skilled in the art that synthetic intermediates, if necessary or desired, can be isolated and / or purified by various well-known techniques, and that various intermediates can often be used directly in subsequent synthetic steps with little or no purification cleaning up. In addition, experienced professionals understand that in some cases, the order of introduction of fragments is not critical. The specific order of the steps necessary to obtain the compound according to the present invention depends on the particular synthesized compound, the starting compound and the relative mobility of the substituted fragments, which is understood by experienced chemists. All substituents, unless otherwise indicated, are as previously defined, and all reagents are well known and accepted in the art.

In this context, the following terms have the indicated meanings: ATP refers to adenosine 5'-triphosphate; BSA refers to bovine serum albumin; DMSO refers to dimethyl sulfoxide; EDTA refers to ethylenediaminetetraacetic acid; EHTC refers to ethylene glycol tetraacetic acid; FBS refers to fetal bovine serum; GFP refers to a green fluorescent protein; HEPES refers to 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid; HWB refers to human whole blood; IC ₅₀ refers to the concentration of a compound that reduces a given response (ligand binding or enzyme response) by 50%; relative IC ₅₀ refers to a relative concentration that provides half of the maximum response to a compound; IVTI refers to inhibition of a target in vivo; JAK refers to Janus kinase; MS refers to mass spectroscopy; NMR refers to nuclear magnetic resonance; NSCLC refers to non-small cell lung cancer; PBS refers to phosphate buffered saline; RNase refers to ribonuclease; room t-ra refers to room temperature; SCLC refers to small cell lung cancer; STAT refers to signal transducers and transcription activators; TED refers to a threshold effective dose; TR-FRET refers to time-resolved fluorescence resonance energy transfer.

Preparatory synthesis 1. (2R) -2- (Trifluoromethyl) oxirane

O ^ CPz

Acetic acid (0.89 ml, 0.052 equiv.) Was added to a solution of (Co ^) - (+) - 1,2-cyclohexanediamino ^^ - bis (3,5-di-tert-butylsalicylidene) cobalt (P) (0) , 90 g, 0.0050 equiv.) In toluene (16.65 ml). Stirred at room temperature for 30 minutes. The solvent was removed in vacuo. Toluene (20 ml) was added and concentrated in vacuo. Cooled to 0 ° C and 2- (trifluoromethyl) oxirane (37.00 g, 330 mmol; ee 80.0%, (2R) is the main enantiomer) was added. It was stirred for 5 minutes and water (0.80 ml, 0.15 equiv.) Was added dropwise. It was allowed to slowly warm to room temperature and was stirred overnight. It was distilled under vacuum at room temperature, collecting the title compound into a chilled flask as a pale yellow oily substance (28.10 g, 76%; ee 99.8%).

¹ H NMR (CDCl ₃ ) δ 2.92-2.94 (m, 1H), 2.98-3.01 (m, 1H), 3.41-3.46 (m, 1H).

The title compound (0.13 g, 1.16 mmol) and methanol (1.3 ml) were mixed. It was cooled to 0 ° C. and triethylamine (0.17 ml, 1.10 equiv.) And thiophenol (0.12 ml, 1.05 equiv.) Were added. Stirred for 30 minutes. GC-MS aliquots showed the formation of 1,1,1-trifluoro-3-phenylsulfanylpropan-2-ol (as a result of ring opening of the title compound); m / z = 222. Chiral LC-MS showed e. 99.8%, (2S) -1,1,1-trifluoro-3-phenylsulfanylpropan-2-ol is the main enantiomer.

Preparatory synthesis 2. 1-Bromo-3,5-difluoro-2-nitrobenzene

Vg

Nitric acid (fuming, 20 ml) was added dropwise to a solution of 1-bromo-3,5-difluorobenzene (35.00 ml, 304 mmol) in sulfuric acid (50 ml) at 0 ° C. It was allowed to slowly warm to room temperature and was stirred overnight. The reaction mixture was poured into a mixture of ice and water (600 ml). Allowed to slowly warm to room temperature. Ethyl acetate (200 ml) and hexanes (100 ml) were added. Stirred until the solid is completely dissolved. Separated layers. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain the title compound as a yellow oily substance (57.37 g, 79%). GC-MS m / z = 237.239 (Br).

Preparatory synthesis 3. 3-Bromo-5-fluoro-Nomethyl-2-nitroaniline

- 5,033,572

sun ₃

NH no ₂

Br

A 2M solution of monomethylamine in tetrahydrofuran (92 ml, 2.00 equiv.) Was added to a solution of 1-bromo 3,5-difluoro-2-nitrobenzene (21.90 g, 92 mmol) in 1,4-dioxane (92 ml). Stir at room temperature for 45 minutes. Water was added and extracted with ethyl acetate. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purified by normal phase chromatography, eluting with a gradient of 20-40% methylene chloride in hexanes, to give the title compound as an orange solid (16.95 g, 74%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 249/251 (M + H).

Preparatory synthesis 4. tert-Butyl- {cis-4- [3-bromo-5- (methylamino) -4-nitrophenoxy] cyclohexyl} carbamate

3-Bromo-5-fluoro-Y-methyl-2-nitroaniline (75.04 g, 301 mmol), tert-butyl- (cis-4 hydroxycyclohexyl) carbamate (89.52 g, 1.38 equiv.) And tetra were mixed (n-butyl) ammonium bisulfate (15.58 g, 0.15 equiv.) in dichloromethane (975 ml) and 5M aqueous sodium hydroxide solution (241 ml). Quickly stirred at 37 ° C in nitrogen atmosphere for five days. Cooled to room temperature. Diluted with dichloromethane (200 ml) and water (400 ml). Separated layers. The aqueous layer was extracted with dichloromethane (3x100 ml). The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purified by normal phase chromatography, eluting with a gradient of 0-40% ethyl acetate in hexanes, to give the title compound as an orange solid (68.57 g, 51%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 442/444 (M-H).

Preparatory synthesis 5. tert-Butyl- {cis-4 - [(4-bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} carbamate

Tert-butyl- {cis-4- [3-bromo-5- (methylamino) -4-nitrophenoxy] cyclohexyl} carbamate (76.92 g, 173 mmol) and 5% platinum on carbon (sulfided, 3.85 g) were mixed ) in tetrahydrofuran (923 ml) in a Parr reactor. Stirred at room temperature under a hydrogen pressure of 414 kPa for three days. Filtered through diatomaceous earth. Washed with tetrahydrofuran. Trimethylorthoformate (165 ml, 8.70 equiv.) Was added to the combined tetrahydrofuran filtrates. Stirred for 22 hours at 63 ° C. The bulk of the reaction was concentrated in vacuo. Diluted with water (400 ml) and ethyl acetate (400 ml). Alkalized with an aqueous solution of sodium carbonate to pH 9. Separated layers. The aqueous layer was extracted with ethyl acetate (2x200 ml). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. It was diluted with methyl tert-butyl ether (400 ml) and sonicated for 30 minutes. Filtered, washed with methyl tert-butyl ether and dried in vacuo to give the title compound as a light brown solid (52.02 g, 71%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 424/426 (M + H).

Preparatory synthesis 6. cis-4 - [(4-Bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexanamine

- 6 033572

Trifluoroacetic acid (666 ml) was slowly added through a dropping funnel to a solution of tert-butyl {cis-4 - [(4-bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} carbamate (222 g, 497 mmol) in dichloromethane (1110 ml) at 0 ° C. It was allowed to slowly warm to room temperature and was stirred overnight. The reaction mixture was concentrated in vacuo. Water (250 ml) was added and made basic with a 50% aqueous sodium hydroxide solution to pH 10. Water (250 ml) was added. It was extracted with a 20% solution of methanol in dichloromethane (1500 ml, then 500 ml, then 250 ml). Wash the combined organic layers with a 2M aqueous sodium hydroxide solution, dry over anhydrous magnesium sulfate, filter and concentrate to give the title compound as a brown solid (155 g, 91%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 324/326 (M + H).

Preparatory synthesis 7. (2R) -3 - ({cis-4 - [(4-Bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} amino) -1,1,1-trifluoropropan-2 ol

(2R) -2- (trifluoromethyl) oxirane (73.29 g, 1.50 equiv.) Was added to a solution of cis-4 - [(4-bromo-1-methyl-1Nbenzimidazol-6-yl) oxy] cyclohexanamine (150 4 g, 436 mmol) in methanol (1053 ml). Stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. Purified by normal phase chromatography, eluting with a gradient of 0-10% ethanol in dichloromethane, to give the title compound as an off-white solid (98.10 g, 52%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 436/438 (M + H).

Preparatory synthesis 8. 3 - ({cis-4 - [(4-Bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} amino) -1,1,1-trifluoropropan-2-ol

Added 2- (trifluoromethyl) oxirane (2.13 ml, 1.02 equiv; ee 80.0%, (2R) is the main enantiomer) to a solution of cis-4 - [(4-bromo-1-methyl -1H-benzimidazol-6-yl) oxy] cyclohexanamine (7.90 g, 24.37 mmol) in isopropanol (130 ml). Heated at 70 ° C. overnight. The reaction mixture was concentrated in vacuo. Purified by normal phase chromatography, eluting with a stepwise gradient from 100% ethyl acetate to 2.5%, up to 5%, up to 7.5%, up to 10% methanol in ethyl acetate, to give the title compound (7.86 g, 74% ) MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 436/438 (M + H).

Preparatory synthesis 9. (5B) -3- {cis-4 - [(4-Bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} -5- (trifluoromethyl) -1,3-oxazolidine- 2-he

- 7 033572

3 - ({cis-4 - [(4-bromo-1-methyl-1H-benzimide azol-6-yl) oxy] cyclohexyl} amino) -1,1,1 trifluoropropan-2-ol (3.34 g, 7.66 mmol; e.i. 80.0%, (2R) is the main enantiomer), 1,1'carbonyldiimidazole (2.48 g, 2.00 equiv.) And 4-dimethylaminopyridine (0.094 g, 0.10 equiv.) in dichloromethane (38.3 ml). Stirred at room temperature under nitrogen overnight. The reaction mixture was concentrated in vacuo. Purified by normal phase chromatography, eluting with a gradient of 05% methanol in dichloromethane to give 3- {cis-4 - [(4-bromo-1-methyl-1H-benzimidazole-6yl) oxy] cyclohexyl} -5- (trifluoromethyl) - 1,3-oxazolidin-2-one (3.28 g; ee 80.0%, (5R) is the main enantiomer).

The product obtained above was partitioned under the following chiral chromatography conditions to give the title compound (0.32 g, 9%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 462/464 (M + H): Enantiomer 1, e. > 99%, 75% / 25% CO ₂ / MeOH, 5 ml / min, 4.6 x 150 mm, Chiralpak AD-H.

Preparatory synthesis 10. (2B) -3 - ({cis-4 - [(4-Bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} amino) -1,1,1-trifluoropropan-2 ol

(5B) -3- {cis-4 - [(4-bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} -5- (trifluoromethyl) -1,3-oxazolidin-2-one was mixed (0.32 g, 0.69 mmol) and potassium trimethylsilanolate (0.36 g, 4.00 equiv.) In tetrahydrofuran (7 ml). Stirred at room temperature under nitrogen for six days. Diluted with water. Filtered, washed with water and dried in vacuo to give the title compound as a white solid (0.22 g, 73%). MS (ER) m / z = ( ⁷⁹ Br / ⁸¹ Br) 436/438 (M + H).

Example 1. (2R) -3 - {[cis-4 - ({4 - [(1,5-Dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

(2K) -3 - ({cis-4 - [(4-bromo-1-methyl-1H-benzimide azol-6-yl) oxy] cyclohexyl} amino) - was mixed

1,1,1-trifluoropropan-2-ol (0.20 g, 0.46 mmol), 1,5-dimethylpyrazol-3-amine (0.056 g, 1.1 eq.), Potassium carbonate (0.158 g, 2 5 eq.), 2- (di-tert-butylphosphino) -2 ', 4', 6'-triisopropyl-3,6-dimethoxy-1,1'-biphenyl (0.046 g, 0.20 eq.), tris (dibenzylideneacetone) dipalladium (0) (0.042 g, 0.10 eq) and acetic acid (0.01 ml) in tert-butyl alcohol (5 ml). They closed it with a screw cap. Heated in a microwave reactor at 120 ° C for 45 minutes. The reaction mixture was concentrated in vacuo. Purified by normal phase chromatography, eluting with a stepwise gradient from 100% ethyl acetate to 1%, to 2.5%, to 5% methanol in ethyl acetate, to give the title compound (0.071 g, 33%). MS (ER) m / z =

- 8 033572

467 (M + H).

Alternative Example 1. (2K) -3 - {[cis-4 - ({4 - [(1,5-Dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6-yl} oxy ) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol

Hn

TU ^with n,

CH ₃

(2I) -3 - ({cis-4 - [(4-bromo-1-methyl-1H-benzimide azol-6-yl) oxy] cyclohexyl} amino) - was mixed

1,1,1-trifluoropropan-2-ol (50 g, 115 mmol), 1,5-dimethylpyrazol-3-amine (18.25 g, 1.43 equiv.) And potassium carbonate (50 g, 3.16 equiv.) in 2-methylbutan-2-ol (400 ml). They were mixed and degassed using a nitrogen sparging line for 15 minutes. 2- (Dicyclohexylphosphino) -3,6-dimethoxy-2 ', 4', 6'triisopropyl-1,1'-biphenyl (2.20 g, 0.034 equiv.) And tris (dibenzylideneacetone) dipalladium (0) (1) were added 60 g, 0.015 equiv.). They were mixed and degassed using a nitrogen sparging line for 5 minutes. Acetic acid (3 ml) was added. Refluxed under nitrogen for 20 hours. 2- (Dicyclohexylphosphino) -3,6-dimethoxy-2 ', 4', 6'-triisopropyl-1,1'-biphenyl (1.10 g, 0.017 eq.) And Tris (dibenzylideneacetone) dipalladium (0) (0.80 g, 0.0075 eq.). He was heated to boiling under reflux in a nitrogen atmosphere for 3 hours. The reaction mixture was concentrated in vacuo. Diluted with water (500 ml). Acidified with a 35% aqueous hydrochloric acid solution to pH 1. Ethyl acetate (100 ml) was added and stirred for 5 minutes. The stirred mixture was treated with activated charcoal (5 g) and filtered through diatomaceous earth. Separated the layers and discarded the organic layer. The aqueous layer was basified with 30% w / w. an aqueous solution of ammonium hydroxide to a pH of 10. Filtered to obtain a solid. Purified by normal phase chromatography, eluting with a 10% mixture of a 2M solution of ammonia in methanol in dichloromethane to give the title compound (52 g, 95%). MS (ER) m / z = 467 (M + H).

Second Alternative Example 1. (2I) -3 - {[cis-4 - ({4 - [(1,5-Dimethyl-1H-pyrazol-3-yl) amino] -1methyl-1H-benzimidazol-6-yl} hydroxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol, crystalline form I.

A. Part of the product obtained by normal phase chromatography in Alternative Example 1 (1.60 g) was dissolved in a 9: 1 mixture of acetone: water (45 ml). It was stirred at room temperature for 15 minutes and SiliaBond® DMT (0.42 g) was added. Filtered after 5 hours at room temperature. The filtrate was concentrated to remove all acetone, then diluted with water (10 ml). It was filtered and dried in vacuo to give a crystalline substance (1.37 g). MS (ER) m / z = 467 (M + H).

C. The product obtained by normal phase chromatography in Alternative Example 1 (47.0 g) in isopropanol (1.15 L) was suspended. The mixture was heated to boiling under reflux to obtain a solution. Vitreous carbon (6 g) was added. After 1 h of reflux, it was filtered through diatomaceous earth. Washed with isopropanol (50 ml) and introduced into the filtrate the seed of the crystalline substance obtained in subsection A, directly above (0.20 g, in parts). It was stirred and allowed to cool to room temperature for 2 hours. It was filtered and dried in vacuo to give a crystalline substance (37.8 g). MS (ER) m / z = 467 (M + H).

Example 2. ^) - 3 - {[cis-4 - ({4 - [(1,5-Dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6yl} oxy) cyclohexyl ] amino} -1,1,1-trifluoropropan-2-ol

- 9 033572

(2B) -3 - ({cis-4 - [(4-bromo-1-methyl-1H-benzimidazol-6-yl) oxy] cyclohexyl} amino) - was mixed

1,1,1-trifluoropropan-2-ol (0.077 g, 0.18 mmol), 1,5-dimethylpyrazol-3-amine (0.030 g, 1.45 equiv.), Potassium carbonate (0.060 g, 2.46 equiv.), 2- (di-tert-butylphosphino) -2 ', 4', 6'-triisopropyl-3,6-dimethoxy-1,1'-biphenyl (0.022 g, 0.25 equiv.), tris ( dibenzylideneacetone) dipalladium (0) (0.0080 g, 0.050 equiv.) and acetic acid (0.01 ml) in tert-butyl alcohol (2.5 ml). They closed it with a screw cap. Heated at 95 ° C. overnight. Diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate was concentrated in vacuo. Purified by normal phase chromatography, eluting with a gradient of 5-100% B (A: dichloromethane; B: 15% mixture of a 0.75M solution of ammonia in methanol in dichloromethane). Further purified by reverse phase chromatography, eluting with a gradient of 5-100% B (A: 10 nM aqueous solution of ammonium bicarbonate with 10% methanol; B: acetonitrile). The pure fractions from ethanol were concentrated, then concentrated again from dichloromethane to give the title compound (0.054 g, 65%). MS (ER) m / z = 467 (M + H).

Example 3. (2R) -3- {[cis-4 - ({4 - [(1,5-Dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6yl} oxy) cyclohexyl] amino} -1,1,1-trifluoropropan-2-ol; dimethanesulfonic acid

(2R) -3 - {[cis-4 - ({4 - [(1,5-dimethyl-1H-pyrazol-3-yl) amino] -1-methyl-1H-benzimidazole-6yl} oxy) cyclohexyl] was mixed amino} -1,1,1-trifluoropropan-2-ol (1.00 g, 2.15 mmol) and acetone (35 ml). Heated at 51 ° C and a solution of methanesulfonic acid (0.30 ml, 2.10 equiv.) In acetone (5 ml) was added dropwise. It was stirred at 51 ° C for 1 h, and then cooled to room temperature. The resulting solid was filtered and dried in a vacuum oven at 70 ° C. overnight to give the title compound (1.25 g, 88%).

X-ray powder diffraction.

The RPD diagrams of crystalline solids were obtained on a Bruker D4 Endeavor X-ray powder diffractometer equipped with a CuKa source (λ = 1.54060 A) and a Vantec detector operating at 35 kV and 50 mA. The sample was scanned from 4 to 40 ° 2Θ with a step of 0.009 ° 2Θ and a scanning speed of 0.5 s / step and with a divergence of 0.6 mm, with a fixed 5.28 mm anti-scattering and 9.5 mm detection slots. The dry powder was packaged in a quartz sample holder and provided a smooth surface using a glass slide. Crystalline diffraction patterns were obtained at ambient temperature and relative humidity. In the field of crystallography, it is well known that for any given crystalline form, the relative intensities of diffraction peaks may vary due to the preferred orientation due to factors such as morphology and crystal shape. In the presence of the preferred orientation effect, the peak intensities are variable, but the positions of the characteristic polymorph peaks do not change. See, for example, US Pharmacopeia No. 23, National Form No. 18, pp. 1843-1844, 1995. Furthermore, it is also well known in the field of crystallography that for any given crystalline form, the angular positions of the peaks may vary slightly. For example, peak positions may shift

- 10 033572 due to changes in temperature or humidity at which the sample is analyzed, due to sample displacement or the presence or absence of an internal standard. In the presented case, the peak position variability of ± 0.2 ° 2θ takes into account the indicated possible deviations, without interfering with the exact determination of the indicated crystalline form. The confirmation of the crystalline form can be based on any unique combination of characteristic peaks (in units of ° 2θ), usually more pronounced peaks. Crystalline diffraction patterns obtained at ambient temperature and relative humidity were corrected according to standard NIST 675 peaks at 8.853 ° and 26.774 ° 2θ.

The characteristics of the obtained sample of the compound from the second alternative example 1 were obtained using the RPD diagram using CuKa radiation having diffraction peaks (2θ values) presented in the table below. 1, and in particular having peaks at 19.5 ° in combination with one or more peaks selected from the group consisting of 11.9 °, 15.4 ° and 17.6 °; with a tolerance of diffraction angles of 0.2 °.

Table 1

Peaks of X-ray powder diffraction of the second alternative example 1

Peak Angle (° 2-theta) +/- 0.2 ° Relative intensity (% of the most intense peak) 1 6.4 17.0% 2 P, 9 46.6% 3 14.7 11.7% 4 15.4 41.9% 5 16,2 27.5% 6 16.9 24.9% 7 17.6 40.1% 8 19.5 100.0% 9 20.8 32.1% 10 21.5 19.6%

The characteristics of the obtained sample of the compound from example 3 were obtained using the RPD diagram using CuKa radiation having diffraction peaks (2θ values) shown in the table below. 2, and in particular having peaks at 21.7 ° in combination with one or more peaks selected from the group consisting of 21.2 °, 18.0 ° and 15.7 °; with a tolerance of diffraction angles of 0.2 °.

Table 2 Peaks of powder x-ray diffraction of example 3

Peak Angle ( ^s ' 2-theta) +/- 0.2 ° Relative intensity (% of the most intense peak) 1 5,6 26.2% 2 10.0 25.7% 3 13.1 28.7% 4 15.7 70.0% 5 18.0 90.5% 6 19.6 64.5% 7 20.6 41.8? Ώ 8 21,2 99.3? / " 9 21.7 100.0% 10 22.3 59.9%

Enzyme assays JAK1, JAK2 and JAK3 in vitro.

To determine the ability of the test compounds to inhibit the activity of JAK1, JAK2, and JAK3 kinases, JAK LanthaScreen ™ kinase analysis (Invitrogen) was used. In these TR-FRET assay formats, an antibody labeled with a long half-life terbium isotope is used as donor particles, and GFP-STAT1 as acceptor particles. To control kin activity

- 11 033572 JAKs used the TR-FRET ratio, where an increase in the phosphorylation of GFP-STAT1 leads to an increase in the TR-FRET ratio. The kinase reaction was carried out in a reaction volume of 12.5 μl on a fine black 384-well Proxiplate plate. To obtain the final reaction conditions, 50 ml of HEPES reagents with pH, 1.76 mm Triton X-100, ATP (20.0 μM for JAK1 and JAK3 enzyme assays or 5 μM for JAK2 enzyme assays), 10.0 mm MgCl _{2 were} added. 1 mM EGTA and 0.01% Brij-35, 0.05 mM GFP-STAT1, 14 nM JAK1 enzyme for JAK1 enzyme assays, 1.0 nM for JAK2 enzyme assays, or 2.5 nM for JAK3 enzyme assays and 4% DMSO and used serial dilutions of the test compounds (diluted 1: 3 from 20,000 to 1 nM). After the addition of ATP / GFP-STAT1, the assay plates were centrifuged for 1 min at a speed of 1000 rpm. The plates were left to incubate at room temperature for 60 minutes, and then 12.5 μl of stopping buffer containing 20 mM EDTA, 2 nM terbium-labeled antibodies against phosphorylated signal transducers and transcription activators [phosphorylated amino acid tyrosine 701] (Tb-anti-pSTAT1) was added. ) [pTyr701], 0.67 mM Tris (hydroxymethyl) aminoethane hydrochloride (Trizma®) with a pH of 7.5, 0.02% NaN ₃ and 0.01% nonylphenyl polyethylene glycol (Nonidet® P40). They were incubated at room temperature for 90 min and read on an EnVision plate reader with an excitation wavelength filter of 340 nm and radiation wavelength filters of 520 and 495 nm. The ratio was determined by the radiation wavelength for GFP-STAT1, which was measured at 520 nm, relative to the radiation at 495 nm for (Tb-anti-pSTAT1) [pTyr701]. For each compound, an IC ₅₀ value was determined using percent inhibition data calculated from the reaction relative to control samples on a plate (active enzyme relative to an enzyme inhibited by tofacitinib at a concentration of 2.0 mM). ACTIVITYBASE 4.0 was used to approximate percent inhibition data at ten concentrations of the compound to the four-parameter logistic equation.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed an IC50 value for JAK1 of 4.0 ± 0.8 nM (n = 6), an IC ₅₀ for JAK2 557 ± 337 nM (n = 6), and an IC ₅₀ for JAK3 of 1910 ± 786 nM ( n = 6). The results obtained demonstrate that the compound of Example 1 inhibits the in vitro JAK1 enzyme. The results also demonstrate that the compound of Example 1 is a more effective inhibitor of the JAK1 enzyme and is selective compared to JAK2 and JAK3 in vitro.

Analysis of proliferation of clone 12 BaF3 with mutant JAK-1 (S729C).

The purpose of this analysis was to determine the inhibitory activity of specific JAK-1 inhibitors using the proliferation analysis of clone 12 BaF3 with mutant JAK-1 (S729C).

The cell line expressing mtJAK1 (S729C) was created by transducing Ba / F3 cells (mouse pro-B cells) with a retrovirus expressing the human mtJAK1 (S729C) -pQCXIN vector DNA. One cell cloning was performed to select Ba / F3 cells expressing the maximum concentration of mtJAK1 (S729C) by serial dilution. A cell suspension of BaF3 with mutant JAK-1 (S729C) was grown and maintained in medium (Gibco RPMI1640, cat. No. A10491-01) containing 10% Hi-FBS (Hyclone, cat. No. 10082-047), 1 μg / ml puromycin dihydrochloride (Sigma, cat. No. 9620), 1.0 mg / ml G418 sulfate (Corning, ref. No. 30-234-CI), hereinafter referred to as R10 +.

Proliferation assays were performed in a culture medium without selection, which is hereinafter referred to as (R10-). Briefly, the grown cells were decanted, centrifuged, and reconstituted in 10 ml of R10-, read using a Beckman Coulter Vi-Cell. Cells were further diluted to 2e4 / ml in R10- and placed on 96-well white matte assay plates (Costar, Cat. No. 3610) at a concentration of 1x10 ³ cells (50 μl) per well. Test compounds were diluted in 100% DMSO in appropriate 96-well polypropylene plates, then further diluted in R10 to give a 2X matrix of test compound. Diluted test compounds were added to the appropriate cell plate to give a CRC of 20 to 0.001 μM. Minimum and maximum control samples were included in each plate. Staurosporin (final concentration 1 μM) was used as the minimum control, and DMSO containing R10 - in the same amount as CRC was used as the maximum control. The plates were coated with water-filled Microcline evaporator caps (Cat. No. LLS-0310) and incubated at 37 ° C in 5% CO ₂ for 3 days. After 3 days of incubation, the plates were removed from the incubator and allowed to cool to room temperature. After cooling, the tablets were developed by adding 100 μl of Cell Titer Glo (Promega, Cat. No. G7571), mixed for 2 minutes on a tablet mixer and incubated for another 10 minutes at room temperature. The luminescence was then read on a Perkin Elmer Victor2 instrument using a memory program for reading luminescence from 0.1 s. IC ₅₀ curves were plotted using the STATS TMAG internal program and GraphPad Prism version 4.03.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of example 1 showed an IC50 value of 956 ± 217

- 12,033,572 nM (n = 4). The result demonstrates that the compound of Example 1 is active against mtJAKl (S729C) expressed in Ba / F3 cells.

Cell analysis of p-STAT3- (p-Tyr705) -IL6-TF-1-JAK1 using AlphaScreen SureFire protocol.

The JAK1 cell assay described below was used to determine the efficacy of the test compounds against JAK1 in cells.

Getting cells.

Cells were kept TF-1 in DMEM medium with 0.5% 26400 (FBS) and 1X Pen / Strep at 37 ° C. 100 thousand cells per well were placed in 96-well black transparent BD plates. The plates were held at room temperature for 30-60 minutes, then incubated overnight at 37 ° C and 5% CO ₂ . Cells were counted using a Vi-Cell counter using a cell suspension at a concentration of 100 cells / ml, and placed 100 μl per well in Beckman Dickinson Biocoat plates (Cat. No. 354640).

Preparation of test compound and processing.

Serial dilutions of compounds 1: 3 in DMSO were obtained and further diluted in this medium. The compounds were studied in the range of 10 concentration points from 20,000 to 1 nM. The diluted compound was added to the appropriate cell plates. The plates were incubated at 37 ° C for 20 minutes. A solution of IL6 at a final concentration of 30 ng / ml was added to the appropriate cell plates and continued to incubate at 37 ° C for 30 minutes. The medium was removed and 50 μl of 1x lysis buffer was added to each well.

PSTAT3 detection.

The following steps were performed in succession: an acceptor mixture was obtained (activating buffer / reaction buffer / acceptor beads): 4 μl of lysate was transferred from 96-well plates to 384-well Proxiplate plates; 5 μl of the acceptor mixture was added to the 384-well Proxiplate plate (s) and the plates were covered with an aluminum lid; shaken for 1-2 minutes on a tablet shaker; the plate was incubated at room temperature for 2 hours with gentle shaking; received a donor mixture (donor granules in dilution buffer); 2 μl of the donor mixture was added to assay plates; closed the tablets with an aluminum lid; shaken for 1-2 minutes on a tablet shaker; incubated at room temperature for 2 hours with gentle shaking; reading tablets using Envision; AlphaScreen Surefire 384 protocol.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of example 1 showed an IC ₅₀ value of 87 nM ± 75 (n = 10). The result demonstrates that the compound of Example 1 inhibits the JAK1 enzyme in a cell assay.

Cell analysis of H1975-JAK1 according to ACUMEN protocol for phospho-STAT3 (Tyr705).

The H1975-JAK1 cell assay was used to confirm the efficacy of the test compounds in NSCLC cells in which the STAT3 pathway is activated by the IL-6 autocrine loop.

Getting cells.

The following stages were sequentially performed: the cells were examined under a microscope; aspirated the medium from the cells using a sterile vacuum pipette; washed cells using approximately 5 ml of PBS; aspirated with PBS using a sterile vacuum pipette; 5 ml of a 0.25% trypsin-EDTA solution was added to a 150 cm ² flask; the flask was gently rocked to cover the cells with trypsin and left to stand under the hood for 3 minutes or the flask was placed in the incubator for 3 minutes to separate the cells from the flask; shaken the flask several times to separate the cells; 10 ml of culture medium was added to the flask, the suspension was carefully distributed on the side of the flask where the cell growth occurred, and mixed in a pipette to grind the cells; centrifuged (1300 rpm for 5 min), re-suspended in 10 ml of culture medium; filtered through a cell filter (BD Falcon 352350, cell filter 70 μm); cells were collected in a 50 ml sterile conical tube; cells were counted using a Vi-Cell counter (0.5 ml cells).

Day 1. Put 30,000 cells per well in 96-well black transparent bottom BD plates. Cells were counted using a Vi-Cell counter, a cell suspension of 300,000 cells / ml was used, and 100 μl / well were placed on Beckman Dickinson Biocoat plates, cat. No. 354640. The plates were maintained at room temperature for 30-60 minutes, then they were placed in an incubator. Then incubated overnight at 37 ° C and 5% CO ₂ .

Preparation of compound and processing.

A deep well plate was prepared containing 1 ml of FBS-free medium with 0.6% DMSO, then 2 μl of compound (10 mM solution in DMSO) was added to obtain a tablet with the original 20 μM solution. Serial dilutions of 1: 3 compounds were performed in FBS-free medium with 0.6% DMSO on dilution plates. The compounds were studied in the range of 10 concentration points from 20 μM to 1 nM. The medium was removed from cell assay plates. Transferred 100 μl of the compounds from the tablet for dilution into assay plates (Beckman Dickinson Biocoat plates, Cat. No. 356440) containing cells bound to 100 μl of medium and used the TEMO program: cell analysis

- 13 033572 of sample transfer using a slow Gem 3797 dispenser. Then the cells were incubated for 4 hours at 37 ° C.

PSTAT3 detection.

On day 1, the following stages were successively carried out: medium was removed; 100 μl of 3.7% paraformaldehyde was added; incubated for 30 min in the dark; washed with 100 μl PBS; 100 μl of cold methanol was added; incubated for 15 min in the dark; washed 2 times using 100 μl of PBS; 100 μl blocking solution (1% BSA in PBS) was added; incubated for 30 min in the dark; 50 μl of primary antibody pSTAT3 1: 500 was added (in blocking solution: 1% BSA; mouse antiphospho-STAT3 (Tyr705)); the plates were covered with aluminum foil and incubated at 4 ° C with gentle shaking overnight.

On day 2, the following steps were performed sequentially: 2 times washed with 100 μl PBS; 50 μl of a secondary antibody (1: 1000; Ab2 ° goat anti-mouse IgG) was added; incubated for 1 h at room temperature in the dark; Washed 2 times with 100 μl of PBS; 100 μl of propidium iodide (1: 1000 in PBS from a commercially available solution) containing RNase (50 μg / ml in PBS) was added; the plates were covered with a transparent film and incubated at room temperature for 2 hours before reading; read on an Acumen Explorer instrument (parameters were adjusted based on the positive and negative wells). Laser (1 scan) 488 nm.

Data processing.

The data was processed using the activity base and analyzed using a 4-parameter non-linear logistic equation (four-parameter logistic curve of the dependence of response on concentration)

Y = lower + [(bottom-top) / 1 + (x / 1C50) at goal] where Y =% inhibition, X = concentration providing% inhibition, bottom. = minimum value of y on the curve, top. = the maximum value of y on the curve, and angle = the steepness of the curve at IC50.

% Ing. = [(average Max- x / average Max - average Min)] 100

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed an IC ₅₀ value of 297 nM (n = 1). The result demonstrates that the compound of Example 1 is active against JAK1 in NSCLC cells in which the STAT3 pathway is activated by the IL-6 autocrine loop.

Cell analysis of P-STAT5 (p-Tyr694 / 699) -EPO-JAK2 using AlphaScreen SureFire protocol.

The selectivity of the studied compounds with respect to JAK1 compared to JAK2 was determined using cell analysis, in which EPO was used to activate the JAK2-STAT5 pathway, and pSTAT5 was used as a read parameter.

Getting cells.

UT-7 cells were kept overnight in DMEM with 0.5% 1600 (FBS) and 1X Pen / Strep at 37 ° C (without GM-CSF). 50,000 cells per well were placed on 96-well black transparent bottom BD plates (in medium without FBS and GM-CSF). The plates were held at room temperature for 30-60 minutes, then incubated overnight at 37 ° C and 5% CO ₂ . Cells were counted using a Vi-Cell counter using a cell suspension at a concentration of 500,000 cells / ml, and 100 μl per well were placed in Beckman Dickinson Biocoat Cat. No. 354640.

Dilution of the compound and treatment.

The compounds were studied in the range of 10 concentration points from 20,000 to 1 nM. Serial dilutions of compounds 1: 3 in DMSO were obtained and further diluted in this medium. The diluted compound was added to the corresponding cell plates and incubated at 37 ° C for 20 min, then 20 μl of EPO (295 ng / ml, final concentration 45 ng / ml) was added to the corresponding cell plates, and incubation was continued at 37 ° C for 30 min

Cell lysis.

The medium was discarded (carefully) and 50 μl of 1X lysis buffer was added to each well (lysate was frozen overnight), (lysis buffer: 5X; diluted in water to a final concentration of 1X).

P-STAT5 detection.

The following stages were carried out sequentially: an acceptor mixture was obtained (activating buffer / reaction buffer / acceptor granules); transferred 4 μl of the lysate from 96-well plates to 384-well Proxiplate plates; 5 μl of the acceptor mixture was added to the 384-well Proxiplate plate (s) using a Multidrop Combi dispenser and the plates were covered with an aluminum lid; shaken for 1-2 minutes on a tablet shaker; the plate was incubated at room temperature for 2 hours with gentle shaking; received a donor mixture (donor granules in dilution buffer); 2 μl of the donor mixture was added to assay plates using a Multidrop Combi dispenser; closed the tablets with an aluminum lid; shaken for 1-2 minutes on a tablet shaker; incubated at room temperature for 2 hours with gentle shaking; ni

- 14,033,572 tablets were washed using Envision; AlphaScreen Surefire 384 protocol.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed an IC _{5u value of} 11.8 ± 5.4 μM (n = 5). The result demonstrates that the compound of Example 1 is selective for JAK1 compared to JAK2 in a cell assay using EPO to activate the JAK2-STAT5 pathway.

Human whole blood tests.

Determination of pSTAT3 (JAK1) and pSTAT5 (JAK2) in lymphocytes and monocytes.

Human Whole Blood Assays (HWBs) were developed and validated to determine the selectivity of the test compounds for JAK1 and JAK2.

Dilute test compounds (1: 3) in 100% DMSO to give 10 points, and then in PBS + 0.1% BSA. Tofacitinib was used as a reference compound in each plate, as well as the maximum signal (stimulated wells) and minimum signal (absence of stimulated wells) to normalize the data. Received an HWB pool from 4 different healthy donors. Blood was placed in a 96-well plate using Tecan Evo 96w and incubated with the test compounds for 1 h at room temperature. After the indicated incubation time, HWB was stimulated using IL6 (206-IL, R&D System) and GM-CSF (PHC2015, Life Technologies) for the next 15 minutes. Dye was added to determine the viability (65-0865, eBiosicience) (1: 1000) using Tecan Evo 96w (5xmix).

The final concentrations in the analysis were as follows: 100 μM compounds, 50 μM tofacitinib, 0.1 μg / ml IL6, 0.038 μg / ml GM-CSF and 1% DMSO. Lysed and fixed HWB using lysis / fixing buffer (558049, Becton Dickinson), adding 900 μl of lysis buffer using Tecan Evo 96w (mixed 10x at high speed). HWB was incubated in a bath at 37 ° C for 10 min. HWB was centrifuged at 500G for 8 min and the supernatant discarded. Cold methanol was added using Tecan Exo 96w to increase cell permeability. Blood cells were incubated on ice for 30 minutes. After this, 2x cells were washed using staining buffer (554656, Becton Dickinson), centrifuged at 3000 rpm for 2 min, the supernatant was discarded, and the following antibodies were added: anti-human CD4 PE, 1: 100 (12-0048, eBioscience), anti-human CD33 eFluor® 450.1: 50 (48-0337, eBioscience), phospho-STAT5 (Tyr694) (C71E5) rabbit mAb, 1: 100 (Alexa Fluor® 488, conjugate) (3939, Cell Signaling) and phospho-STAT3 (Tyr705) (D3A7) XP ™ rabbit mAb 1: 200, (Alexa Fluor® 647, conjugate) (4324, Cell Signaling). Antibodies were incubated for 1 h in the dark at room temperature, then 2x cells were washed and read on a Macsquant cytometer (Miltenyi Biotec). Data were obtained on CD4 + (lymphocytes) and CD4Low CD33Hi (monocytes) to measure fluorescence in cells expressing pSTAT3 and pSTAT5, respectively. Data was analyzed using FlowJo version 10 and normalized average fluorescence relative to the maximum and minimum signal to determine the values of the IC ₅₀ . Graph Pad prism 5 software was used to plot dose response curves.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed an IC50 value of 2.40 ± 0.64 μM (n = 3) for JAK1 and an IC _{50 of} 13.0 ± 3.2 μM (n = 3) for JAK2. The results obtained demonstrate that the compound of Example 1 shows about 5 times higher efficacy against JAK1 compared to JAK2 in human whole blood analysis.

Analysis of mouse IVTI (JAK1) pSTAT3.

The purpose of this assay was to measure the ability of the test compounds to inhibit STAT3 phosphorylation (STAT3 activation) in a mouse H1975 xenograft model. H1975 cells were grown in accordance with the specification of the American Type Culture Collection (ATCC) with the minimum possible number of transfers. Cells were grown and maintained in RPMI-1640 medium supplemented with 10% FBS and incubated at 37 ° C in 5% CO ₃ . Cells were harvested by standard methods and mixed with a BD Biosciences basement matrix matrix (MATRIGEL®) to achieve a 1: 1 cell to matrix ratio in the cell suspension, with a volume for inoculation of 0.2 ml of cells in a matrix suspension containing 5e6 cells. During the inoculation procedure, the cell suspensions were kept on ice and implantation began within 1 h after harvesting the cell culture. All implants were injected subcutaneously into the right back flank of female nude mice purchased from Harlan. All mice were fed ad libitum with Harlan Teklad No. 2920X and provided with water in gel packs. The implanted tumor cells were allowed to grow to solid tumors and were measured twice a week with body weight starting 5–9 days after implantation. Tumor volume was determined by the equation: 0.536xLxW ^A 2. Upon reaching a tumor volume of about 200-250 mm ³ , animals were randomized using a multitask unit for randomization, and placed in a group that was treated with a vehicle containing 6-10 animals experimental groups containing 6 animals. The studied compounds were formulated in a 1% solution of a SCE vehicle containing 0.25% Tween 80 and 0.05% antifoam agent

- 15,033,572 with 1.1 mol. eq. methanesulfonic acid for in situ salt formation. No acid was added to the control group treated with the vehicle. Doses were calculated based on recent measurements of average weight in the group. Compounds were administered through an oral probe to conduct dose-response studies and to determine the duration of the drug. Studies of the dose-response response were carried out by administering a single dose 2 hours before the extraction, processing and freezing of the tumor. Studies to determine the duration of the drug were performed by administering a single dose equal to TED70, as determined in the dose response study.

Tumor tissue processing.

Tumors were collected and cut to obtain a fragment of about 150-250 m ³ and immediately dropped into a 12x75 mm test tube containing 1 ml of ice lysis MSD Tris buffer (Meso Scale Discovery, Cat. No. R60TX-2) and 1X HALT (Thermo Scientific, Product No. 1861281). Samples were homogenized for approximately 15 seconds using the Omni disposable hard tissue sample tip homogenizer (Omni International, Cat. No. 3_750H) and allowed to stand on wet ice for the next 15-25 minutes, then transferred to a freezer at -60 ° C and kept overnight. Samples were removed from the freezer and allowed to stand at room temperature to initiate thawing. As soon as the samples began to melt, they were transferred to wet ice and continued to thaw. After thawing, the samples were vortexed and transferred into a 1.8 ml microcentrifuge tube. The lysates were centrifuged at 14000Xg for 30-60 minutes at 4 ° C. Lysates (200 μl) were transferred to a 96-well polypropylene plate (Costar, Cat. No. 3879). Protein concentration was determined using a Pierce BCA Protein Assay Kit (Cat. No. 23225) as described below. Briefly, a standard curve was drawn using a BSA standard diluted in RIPA lysis buffer containing 1X HALT to give an operating range of 2000 to 25 μg / ml. All lysates were diluted 1:10 in RIPA lysis buffer containing 1X HALT. 25 μl of standard and experimental samples was pipetted into a 96-well plate (Falcon, Cat. No. 353072), 200 μl of working reagent was added to each well, and the tablet was thoroughly mixed on a tablet shaker for 30 s. The plate was closed and incubated at 37 ° C for 30 minutes. The plate was cooled to room temperature and absorbance was measured at 562 nm or near the indicated value using a Molecular Devices Spectra Max. Protein concentrations for each sample were determined using SoftMax Pro 6.3 software. After quantitative analysis of the tumor images, lysates were frozen at -80 ° C in a 96-well polypropylene plate and analyzed later.

PSTAT3 measurement.

Analysis of pSTAT3 (Tyr705) was performed as follows.

The pSTAT3 MSD assay kit from Meso Scale Discovery (Cat. No. K150DID-2) was used. Instead of a set of phosphatase and protease inhibitors, 100X HALT protease and phosphatase inhibitors can be used based on ease of use and operation. The Tris MSD lysis buffer was prepared by adding 100X HALT protease inhibitor and phosphatase inhibitor to 1X final concentration, which was designated as complete MSD lysis buffer. Tumor samples were removed from the freezer at -80 ° C and allowed to stand at room temperature to initiate thawing. During thawing of samples, a storage plate with pSTAT3 Tyr705 (cat. No. K150DID-2) was blocked with 150 μl of blocking solution for at least 1 h at room temperature on a tablet shaker with vigorous shaking (300-1000 rpm). Before shaking, the plates were closed with the adhesive cover of the tablet. The blocking solution had a ratio of Blocker A (Cat. No. R93BA-4) 600 mg: 20 ml of 1X MSD Tris Wash Buffer (Cat. No. R61TX-2). As soon as the samples began to melt, they were transferred to wet ice and continued to thaw. Upon completion of thawing, the mixture was lightly mixed in a pipette using a multichannel pipette. The samples were normalized in ice-cold MSD complete lysis buffer to a protein concentration of 0.4 μg / ml X 100 μl. All normalized tumor samples were kept on ice in a 96-well polypropylene plate until they were added to the pSTAT3 storage plate. After blocking the accumulation plate, pSTAT3 was washed 4X using 250-300 μl of 1X MSD Tris wash buffer using Thermo Labsystems Multidrop 384 plate washer. After the last wash, the plate was tapped lightly to remove the remaining wash buffer. A total volume of 25 μl (10 μg) of normalized tumor lysate per well was added and incubated for another 2–3 hours at room temperature on a plate shaker with vigorous shaking (300-1000 rpm). During the incubation, the antiphospho-STAT 3 detection antibody labeled with sulfo-TAG, which is presented in the indicated set in the antibody dilution buffer (1 ml of blocking solution mixed with 2 ml of 1X MSD wash buffer) was diluted by adding 60 μl of antiphospho-STAT3 detection antibody labeled with sulfo-TAG to 2.94 ml of antibody dilution buffer. The resulting antibody solution was kept on wet ice until use. After incubation of the lysate, the tumors were washed with a 4X plate using 250-300 μl of 1X Tris MSD wash buffer. After the last wash, a plate was tapped to remove the remaining wash buffer, and 25 μl / well of anti-detector was added.

- 16,033,572 antiphospho-BTATZ bodies labeled Sulfo-TAG. The plate was incubated for another 1 h at room temperature on a tablet shaker with vigorous shaking (300-1000 rpm). During incubation, the 4X T MSD buffer buffer with surfactant (Cat. No. R92TC-3) was diluted to 1X using deionized water and kept at room temperature. Upon completion of incubation, the detection antibody was washed with a 4X plate using 250-300 μl of 1X Tris MSD Wash Buffer. After the last wash, the plate was lightly tapped to remove the remaining wash buffer, and a total volume of 150 μl / well of 1X MSD T-Surfactant Read Buffer was added. The plate was read on a Meso Quick Plex SQ 120 instrument. Data was analyzed as described below.

Calculation of percent inhibition of pSTAT3.

The original tablet data from the Meso Quick Plex SQ 120 was copied and pasted directly onto a Microsoft Excel 2010 worksheet. We systematized the data in a suitable format (dose response or duration), copied and pasted directly into JMP version 11 to calculate the percent inhibition of pSTAT3 (see the formula below).

| [1 - (signal of the processed sample / average signal of the control sample with the carrier)] * 100.

The average value in the experimental group was determined using one-way analysis of variance and compared with the average value in the control group with the carrier using the Dunnett test.

TED calculations.

The TED ₅₀ and TEC ₅₀ values were determined based on a dose-response study. TED ₅₀ is the dose required to achieve 50% inhibition of pSTAT3, and TEC ₅₀ is the plasma concentration to achieve 50% inhibition of pSTAT3, respectively, after 2 hours.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed a TED ₅₀ 3 of 5 mg / kg and a TEC _{50 of} 11.1 μM (n = 1). The results demonstrate that the compound of Example 1 effectively inhibits JAK1-STAT3 signaling (using pSTAT3 as a read parameter) in vivo 2 hours after an oral dose. The results obtained demonstrate that the compound of Example 1 also has PK / PD activity correlating with TEC ₅₀ .

Rat IVTI (JAK1) pSTAT3 assay.

The purpose of this assay was to measure the ability of the test compounds to inhibit pSTAT3 expression in the rat H1975 xenograft model. H1975 cells were grown in accordance with the specification of the American type culture collection with the minimum possible number of transfers. Cells were grown and maintained in RPMI-1640 medium supplemented with 10% FBS and incubated at 37 ° C in 5% CO ₂ . Cells were collected by standard methods and mixed with a BD Biosciences basement matrix matrix (MATRIGEL®) to achieve a cell to matrix ratio of 1: 1, with a volume for inoculation of 0.2 ml of cells in a matrix suspension containing 2e6 cells. During the inoculation procedure, the cell suspensions were kept on ice and implantation was started within 1 h after cell culture collection. All implants were injected subcutaneously into the right posterior flank of female NIH hairless rats purchased from Taconic. All rats were fed ad libitum with Harlan Teklad Global Diets No. 2920X and provided with bottled water. The implanted tumor cells were allowed to grow to solid tumors and were measured twice a week with body weight. Measurement of body weight and tumor volume was started at 5-9 days after implantation. Tumor volume was determined by the equation 0.536xLxW ^A 2. Upon reaching a tumor volume of about 350-400 mm ³ , animals were randomized using a multitask unit for randomization and placed in a group treated with a vehicle containing 6 animals and in several experimental groups containing 6 animals each. The studied compounds were formulated in a 1% solution of a SCE vehicle containing 0.25% Tween 80 and 0.05% antifoam agent with 1.1 mol eq. methanesulfonic acid for in situ salt formation (1.1 ml of a 1N methanesulfonic acid solution per 1 mg of compound). No acid was added to the control group treated with the vehicle. Doses were calculated based on recent measurements of average weight in the group. All compounds were administered through an oral probe to conduct dose-response studies and to determine the duration of the drug. Studies of the dependence of the response on the dose was carried out by introducing a single dose 2 hours before extraction, processing and freezing of the tumor. Studies to determine the duration of the drug were performed by administering a single dose equal to TED70, as determined in the dose response study. At several time points, tumor removal, processing, and freezing were performed as described below.

Tumor tissue processing.

Tumors were collected and cut to obtain fragments of a size of about 200-250 m ³ and immediately dropped into a 12x75 mm test tube containing 1 ml of ice lysis MSD Tris buffer (Meso Scale Discovery, Cat. No. R60TX-2) and 1X HALT (Thermo Scientific, Product No. 1861281). Homogenized about

- 17 033572 samples for about 15 s using the disposable Omni hard tissue sample homogenizer tip (Omni International, cat. No. 3_750H) and left to stand on wet ice for the next 15-25 minutes, then transferred to the freezer at - 60 ° C and kept overnight. Samples were removed from the freezer and allowed to stand at room temperature to initiate thawing. As soon as the samples began to melt, they were transferred to wet ice and continued to thaw. After thawing, the samples were vortexed and transferred into a 1.8 ml microcentrifuge tube. The lysates were centrifuged at 14000Xg for 30-60 minutes at 4 ° C. Lysates (200 μl) were transferred to a 96-well polypropylene plate (Costar, Cat. No. 3879) in accordance with the design of the 96-well sample matrix. Protein concentration was determined using the Pierce BCA Protein Analysis Kit (Cat. No. 23225) as described below. Briefly, a standard curve was drawn using a BSA standard diluted in RIPA lysis buffer containing 1X HALT to give an operating range of 2000 to 25 μg / ml. All lysates were diluted 1:10 in RIPA lysis buffer containing 1X HALT. 25 μl of standard and experimental samples was pipetted into a 96 well plate (Falcon, Cat. No. 353072) and 200 μl of working reagent was added to each well and the tablet was thoroughly mixed on a tablet shaker for 30 s. The plate was closed and incubated at 37 ° C for 30 minutes. The plate was cooled to room temperature and absorbance was measured at 562 nm or near the indicated value using a Molecular Devices Spectra Max. Protein concentrations for each sample were determined using SoftMax Pro 6.3 software. After quantitative analysis, tumor images were frozen at -80 ° C in a 96-well polypropylene plate and analyzed later.

PSTAT3 measurement.

Analysis of pSTAT3 (Tyr705) was performed as follows.

The pSTAT3 MSD assay kit from Meso Scale Discovery (Cat. No. K150DID-2) was used. Instead of a set of phosphatase and protease inhibitors, 100X HALT protease and phosphatase inhibitors can be used based on ease of use and operation. Used all other reagents from the set. The Tris MSD lysis buffer was prepared by adding 100X HALT protease inhibitor and phosphatase inhibitor to 1X final concentration (designated as full MSD lysis buffer). Tumor samples were removed from the freezer at -80 ° C and allowed to stand at room temperature to initiate thawing. During thawing of samples, a storage plate with pSTAT3 Tyr705 (cat. No. K150DID-2) was blocked with 150 μl of blocking solution for at least 1 h at room temperature on a tablet shaker with vigorous shaking (300-1000 rpm). Before shaking, the plates were closed with the adhesive cover of the tablet. The blocking solution should have a Blocker A ratio (Cat. No. R93BA-4) 600 mg: 20 ml of 1X Tris MSD Wash Buffer (Cat. No. R61TX-2). As soon as the samples began to melt, they were transferred to wet ice and continued to thaw. Upon completion of thawing, the mixture was lightly mixed in a pipette using a multichannel pipette. The samples were normalized in ice-cold MSD complete lysis buffer to a protein concentration of 0.4 μg / μl X 100 μl. All normalized tumor samples were kept on ice in a 96-well polypropylene plate until they were added to the pSTAT3 storage plate. After blocking the accumulation plate, pSTAT3 was washed 4X using 250-300 μl of 1X MSD Tris wash buffer using Thermo Labsystems Multidrop 384 plate washer. After the last wash, the plate was tapped lightly to remove the remaining wash buffer. A total volume of 25 μl (10 μg) of normalized tumor lysate per well was added and incubated for another 2–3 hours at room temperature on a plate shaker with vigorous shaking (300-1000 rpm). During the incubation, the antiphospho-STAT3 detection antibody labeled with sulfo-TAG, which is presented in the indicated set in the antibody dilution buffer (1 ml of blocking solution mixed with 2 ml of 1X MSD wash buffer) was diluted by adding 60 μl of antiphospho-STAT3 detection antibody with labeled sulfo-TAG to 2.94 ml of antibody dilution buffer. The resulting antibody solution was kept on wet ice until use. After incubation of the lysate, the tumors were washed with a 4X plate using 250-300 μl of 1X Tris MSD wash buffer. After the last wash, the plate was lightly tapped to remove the remaining wash buffer and 25 μl / well of an antiphospho-STAT3 detection antibody labeled SulfoTAG was added. The plate was incubated for another 1 h at room temperature on a tablet shaker with vigorous shaking (300-1000 rpm). During incubation, the 4X T MSD buffer buffer with surfactant (Cat. No. R92TC-3) was diluted to 1X using deionized water and kept at room temperature. Upon completion of incubation, the detection antibody was washed with a 4X plate using 250-300 μl of 1X Tris MSD Wash Buffer. After the last wash, the plate was lightly tapped to remove the remaining wash buffer, and a total volume of 150 μl / well of 1X MSD T-Surfactant Read Buffer was added. The plate was read on a Meso Quick Plex SQ 120 instrument. Data was analyzed as described below.

Calculation of percent inhibition of pSTAT3.

Copy and paste the original tablet data from the Meso Quick Plex SQ 120 directly onto

- 18 033572 worksheet Microsoft Excel version 2010. We systematized data in a suitable format (dose response or duration). Copy and paste directly into the JMP version 11 program to calculate the percent inhibition of pSTAT3 (see formula below).

| [1 - (signal of the processed sample / average signal of the control sample with the carrier)] * 100.

The average value in the experimental group was determined using one-way analysis of variance and compared with the average value in the control group with the carrier using the Dunnett test.

TED calculations.

The TED ₅₀ and TEC ₅₀ values were determined based on a dose-response study. TED ₅₀ is the dose required to achieve 50% inhibition of pSTAT3, and TEC ₅₀ is the plasma concentration necessary to achieve 50% inhibition of pSTAT3, respectively, after 2 hours.

A compound falling within the scope of the present invention was tested in the above assay essentially as described above. The compound of Example 1 showed a TED ₅₀ value of 9 mg / kg and a TEC ₅₀ value of 3.9 μM (n = 1). The results demonstrate that the compound of Example 1 hits a target in the rat model IVTI H1975.

A study of efficacy in a HCC827 tumor xenograft model.

The purpose of this analysis was to measure the ability of the compounds to inhibit tumor growth in a mouse HCC827 xenograft model. HCC827 cells (ATCC No. CRL-2868, lot No. 59392891) were grown in accordance with the specification of the American Type Culture Collection with the minimum possible number of transfers. Cells were grown and maintained in RPMI-1640 medium supplemented with 10% FBS and incubated at 37 ° C in 5% CO ₂ . Cells were collected from flasks using TryPLE, washed twice in DPBS, resuspended in HBSS and mixed with a BD Biosciences basement matrix matrix (MATRIGEL®) to achieve a cell to matrix ratio of 1: 1 in the cell suspension, giving an inoculation volume of 0, 2 ml of cells in a matrix suspension containing 5e6 cells. During the inoculation procedure, the cell suspensions were kept on ice and implantation began within 1 h after harvesting the cell culture. Implants were injected subcutaneously into the right posterior flank of female CB17 SCID mice purchased from Harlan (18-20 g). The mice were fed ad libitum with Harlan Teklad Protein Extruded 2920X and provided access to water in a hanging drinking bowl. The implanted tumor cells were allowed to grow to solid tumors and were measured twice a week with body weight starting from the seventh day after implantation. Tumor volume was determined by the equation: 0.536xLxW ^A 2. Upon reaching the tumor volume of about 150-200 mm ³ animals were randomized and divided into groups containing 5-8 animals. The studied compounds were formulated in a 1% solution of a SCE vehicle containing 0.25% Tween 80 and 0.05% antifoam agent with 1.1 mol eq. methanesulfonic acid for in situ salt formation (1.1 ml of a 1N methanesulfonic acid solution per (insert molecular weight) 1 mg of the compound). The test compound was stored at room temperature. Once a week, the compounds were formulated and stored at room temperature. No acid was added to the control group treated with the vehicle. Doses were calculated based on recent measurements of average weight in the group. Compounds were administered via an oral probe for 28 days according to the BID (twice a day) or QD (once a day) scheme, depending on the dose.

A compound falling within the scope of the present invention was tested in this assay essentially as described above. The compound of Example 1 showed inhibition of tumor growth by 31% at a dose of 30 mg / kg BID, tumor regression by 22% at a dose of 60 mg / kg BID, and tumor regression at a dose of 120 mg / kg QD. The results obtained demonstrate that the compound of Example 1 leads to tumor regression in the HCC827 tumor xenograft model.

Claims (10)

1. The compound selected from the group consisting of F, C .OH NH Hn CH ₃ - 19 033572 or its pharmaceutically acceptable salt and or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, which represents or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 1, which represents 4. A pharmaceutical composition for inhibiting Janus kinase 1 (JAK1) comprising a compound or salt according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, diluent or excipient. 5. The use of a compound or its salt according to any one of claims 1 to 3 for the treatment of cancer by inhibiting JAK1. 6. The use according to claim 5, characterized in that the cancer is selected from the group consisting of lung cancer, including non-small cell lung cancer and lung adenocarcinoma, adenocarcinoma, hepatocellular carcinoma, including hepatocellular carcinoma of Asian residents, colorectal cancer, breast cancer and leukemia, including acute lymphocytic leukemia. 7. The use according to claim 6, characterized in that the cancer is a non-small cell lung cancer. 8. The use according to claim 6, characterized in that the cancer is lung adenocarcinoma. 9. The use according to claim 6, characterized in that the cancer is breast cancer. 10. The use according to claim 6, characterized in that the cancer is hepatocellular carcinoma of the inhabitants of Asia. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2